Observation of body tissues and intracellular ions and molecules is an essential technique used in the field of molecular biology. An optical microscope is mainly used to observe body tissues. A laser confocal microscope is used to observe a part of a living cell by irradiating light to that part while moving the position of irradiation.
Major observation techniques of the optical microscope include a fluorescent observation method. In this method, intracellular ions and molecules of body tissues are colored with a fluorescent dye, an excitation light is irradiated to the fluorescent dye, and then the fluorescent light emitted from the excited fluorescent dye is observed.
Since the wavelengths of the fluorescent light and the excitation light are not identical, intracellular molecules can be detected by analyzing the fluorescent light and the molecular concentrations can be measured by analyzing the fluorescent light. The fluorescent observation has the following advantages:    (i) Objects as small as ions and molecules can be observed;    (ii) Only specific ions and molecules can be observed; and    (iii) Since the intensity of the fluorescent light changes depending on the ionic and molecular concentrations, these concentrations can be measured.
However, the fluorescent observation generally has the following disadvantages:
(i) Excitation light having an ultraviolet wavelength band is harmful to cells; and
(ii) When the fluorescent dye is continuously exposed to the excitation light, the fluorescent dye discolors. This means that the intensity of the fluorescent light decreases.
There is a case that the cell may get out of the microscopic field during observing a moving cell by using a fluorescent microscope capable of fluorescent observation and then the observation is interrupted to continue.
To solve this problem, (1) the field of view is enlarged by decreasing the magnification of the objective lens, (2) cellular movement is suppressed mechanically or chemically, or (3) the stage is moved to allow the cell under observation to come to the center of the field of view. However, the method in (1) decreases spatial resolution, whereas the method in (2) may adversely affect the cell under observation.
Regarding the solution in (3), Patent References 1 and 2 disclose optical microscopes equipped with a function of tracking a microscopic cell under observation. These optical microscopes can control the cell position as well as the observation area and make recordings, using transmission light.
Patent References 3 to 5 disclose fluorescent microscopes capable of controlling the cell position as well as the observation area and make recordings, using fluorescent light.
Non-patent References 1 to 5 describe the method of observing a moving paramecium by fastening it within the field of view of a microscope as a relatively large object to be observed.
Immune system cells having a function of protecting human body from bacteria and viruses is valued as primary importance of cells capable of moving freely within a culture solution in the fields of medicine and biology (Non-patent Reference 6).
There are a number of intracellular molecules affecting our immune function, and it is necessary to use a fluorescent observation to detect those molecules and to measure the temporal change of molecular concentrations.
Immune system cells, which are floating in a solution without attaching to the surface of a slide glass, are classified into floating cells. The floating cells within a petri dish change their positions at all times due to the convection of the culture solution, gravity, and interaction with the wall, bottom of the petri dish, solution surface, and other cells. It is considered that one cell tracking method by Oku et al. described in Non-patent Reference 1 may be used to observe floating cells.